Production of devitaminized casein by solvent extraction



l PatentedJuly 26,

BY SOLYENT EXTRACTION Samuel M. Weisberg; and Joseph Greenspan,

Baltimore, Md., assignors, by mesne `assignments, to National Dairy` Research `Laboratories, Inc., NewlYork, N. Y., a. corporation o! HI-Delaware Application october 18,1945, seal No. 623,146

. Thisinvention `.relates tothe separation of vitamins from a casein, :such as commercial casein. More particularly,` it relates z to `a simple and rapid method fortheproduction oi edible casein substantially free :from all known vitlmans. It especially relates to the production of `socalledvitamin-freebasal diet casein. i

Vitamin free casein has many uses. It is `valuable inlthe lproduction oflbacteriological media.

" It is particularly 1valuableinl the preparation of abasal diet for animal feeding tests, such as `tests made `to determine the effect of` vitamin deficient diets. Various methods havebeen proposed heretofore for the productionjof substantially vitamin free casein.` In generals these methods rare-sub` ject to several disadvantages. Most of them .are

slowf tedious andicumbersome. l Some `of them tend `to alter the -molecular- `structure of the l feeding purposes. i

In general, the methods ploy a; series `oiwashings or` leachingsrsuch as with water `acidiiied withaceticacid, followed byg i alcohol i washes. `Thisgmethod t usually takes from about?! to TabouttlO ,days. Some `methods use' ahighlyconcentrated aqueous-solution o-f a salt-such as sodium chloride, `to leach `out the i vitamins `and` follow thisiwith washings with 1 acetic solutions `and then withhwater;` these rare also slow and cumbersome. Other methodsl involve autoclavinglongperiods of time, or successive solution oit-he casein in alkali and precipitai tion of the casein byacidiiication;` these methods tend to degrade the molecular structure of the casein, `thereby i altering. its value for feeding purposes. Known processes employing an alcohol as theleaching agent are very time-consuming, oftenrequiring as much as two weekscto produce i a suitable so-called vitamin free casein. w.

:Experimental attempts to `prepare ,avitamin free` casein by treating `with a large amount of alkali to substantially disperse the `caseinand extracting with an alcohol; followed by acidifying i thecasein to lower thepH ot the product gave a product Whichfzwas` of; `dark color and which .contained many very `dark yellowish or orange i colored granules;` the llattergave thetproducta very undesirable appearance. Furthermore, thevitamins@were`- destroyedy-in such a treatment; and `were not` recoverable.

process for the production of` a.; substantially vitamin-tree, high quality; basaldiet casein;` i

. i y .I'tlis anwobject.ofitherinvention to provide af` `It is another `object of" theinventionto provide a" simpler, rapid and eilcient 4process for.` the 1. reduction in time separation of vitamins from casein, such as commercial casein.

It is another object of the invention to provide an easily controlled, rapid process for recovering"- Athe vitamins from casein, such as commercial "'casein. i v

Other objects perior produ-ct is obtained. y f v `It has been found ;that,' if dried granular, commercial casein is treated With'an alkaline` agent, such asan alkaline agent in aialcohol solution, `and the pH of the casein brought within the range of about 4.7 to about 6.0 and preferably-f within the range of about 4.8 to about 5.5', that the` granules will become Aswollen but they will.;` not become gelatinous orsoluble, and that if this` swollen granular casein isextracted with aselec tive solvent-such as methanol, thevitamin con# 1 tent is very rapidly.removed,or desorbed-from The alkaline agent treatment` and n v.the extraction may be and preferably are carriedout simultaneously. 4The extract phase which is removed 'from the casein. :may be distilled` to' drive oi the solvent and other vvolatile materials i and the vita-minsmayybe obtained or recovered as ortfrom the still residue. The vaporized solvent may be `recondenseol and re-usedin theAex-lT traction system. If-.the alkaline agent is volatilew it may bedistilled` oi with the solvent and reused the casein.

and if` not may beneutralized and the saltspermitted toremain with the recovered vitamin The processmay beV operated continuously-and4 conveniently in a closedfsystem. 'A highquality 1 substantially vitamin-free 4casein may be obtained -f thereby. in about five hours; this is an enormousy thatiis required by prior art" processes. The new process maybe illustrated by means of the accompanying drawings, but is not limited: thereto. `Figure .1 isa general sketchor ow di1L agram of the process. A'charge of casein ISL-.invA the extractor system I I is contacted with treating e. solvent Aor solvent plusi alkaline.` agent, which enters the extractor system by" means of' inlet ^|8.` `Fresh7 `solvent,`` or alkaline agent, or both `maybe` supplied by ineans of inlet of the invention will become apparent as it is more fully developed hereinafter; -a It has now` been found,-l in accordance with., Athe invention, thatlif .caseiniscontacted` orf. extracted witha selectivesolvent while the` pH of theV casein is controlled lwithin a critical range.` the extractive efiiciency of the solvent is` very` markedly increased and surprisingly indeed, Vthere `gis `no degrading effectonthe casein. Thus,the i extraction timeismarkedly reducedand a su-` I'I. The extract phase passes from the extractor system II, byrneans of line I2, to the still I3. There the solvent is vaporized, and if the alkaline agentis volatile, it, too, is vaporized. The vapors pass, 'by means of line'v IA, -to the' total condenser I5, yWhere they are condensed? the condensate passes by means of line I6 to the inlet I8. The`A` still residue contains the vitamin extract; this 20. The lprocess preferably is carricout-.finla closed system.VY Y e Figure 2 is a sketch of an extractorjsystemior one embodiment of the invention .-in,..wl;ch the casein charge is added as a batch.` The caseinx' is held in a container 2|, which may be a cloth bag or the basket of a centrifuge. The Vtreating solution (i. efsolvent, or sollent'iplus alkaline agent) enters the extractor ILby meansoi. inlet.

I8, and the extract phaseleaves the extract'oroy' means of line I2. Whenfthe extractionis com*n plete, thecharge lof'fcasein -is^rernovec`l;l Y Y Figure 3 is a sketchof an extractor -systemTer'- another'embodiment of the invention 'in which the casein chargeis `continu('ausly added, and the extracted-casein is continuously-removed; VVThe ,treatingsolution (i. insolvent, or solvent plusr alkalineagent) enters ltheextractor-loy means `of inlet '58, r'a'rldl'tlie extract phase leaves thefextractor v21337 "means of5linefl2; 'The casein enters the extracter '-loy means'ofj-inle't v22, andthe exthrough' the esi-tractor by mehaniearmeans,auch

as I'com e'yorbuckets 'or spiralconveyors; the-exe tractormaywbe a centrihigvvith I,ine'elianicall Incensi for advancing 'thec'as'einmcake" from 'the 'Y inlet end to the exit end; n y,

Thel process-of .the invention may be conducted in'vari'ous itypes of extraction equipment. It is wherein the'casein may beheldinfthebasket, Aand l the-slightly alkaline solvent may-:be `passed theref through, boiled, reoondensed,and recycled. '-Afj counter-current process, in whichv :the'substrate 145 adaptable for use with V'an enclosed lcentrifuge casein and the vsolvent vare lpassed "t'hrough an extractor 'indifferent 'directions, may be employed; Ain this 'the' raffinate y(ilevitaniinized v casein) will be vconti uous'lyjremoved-- at yone zone,

1 .interrelated asl will be understood by those Yskilled in the .'herein. j

art in View of the explanation "Byfemploying a solventnsuch as methanol and an Yalkaline agent such. as Yammonia as the treating agent, a particularly fast and complete exl YTtnaction.may'be accomplished. AThe higher the pH.' 'themorefrapidis the Vextraction. Usually a treating scli'tionlliiin the range of about '7 to about Stat the start of the casein treatment will beancptiniumlvalue. At the end of the treatmentgorextraction the pH of the casein should `not'beabove about 6.0. The exact gure will depend on the initial acidity of the casein, its hardness, method of manufacture-,the 'source of the milk and other 4factors'. general the'lnal pH will not be above about'one1unit-above`the! iso-electric point ofthe/casein (4556 51:04.65). Asif a practical matter `:thersH' must lnot be :abovetiiat Whichwillcausethe casein to gelatinizegdisperse or lump. The individualiinitiairlparticles.should remain as such aiter''the treatment so thatitiiey vmay leertreaited andhandled iin-ithe-usnal manner?.v i This -is to ber'distinguished'from `priorl art xprocesses ira-Which the casein is dispersed Withan alkalijlconnected `to la caseina'te) and thenrepre Y Ycipit'ated'vvirtl'ian 'acidas casein V1reza\`7i`1'ig-"a part' oi' fthevitarr1in1in'the motheriliquid. VThe pH may be 'determined'fconvenientiy loy` the glass 'electrode pH meter.

'rheyfouowing exemplars mended order to.

illustrate'andtpoint out somefof the advantages of' the invention, but lisin no :wise to be cim'struerll as Va limitation of the invention, asf'ctlierw'isedisu 1 Y closed andclaime-'hereinf movedfat'another zone ofA the extractor. Y

AA selective solvent-for the vitamins is used;

the lower alcohols-containing from l Yto 5 carbon atomsV molecule-are' suitable, and of these,

metharn'llv is preferred. -Otherselective solvents ineludeik'etones suchas`-methyl isobutyl ketone,

and-"ethers Ysuch as dipropylether. The alcohol may 'be `used fin -'a strength lnthe range offab'out 196% tof aboutj80%, fby volume.' Various .-rnaterials maybe used as Vthe alkaline agents.` e. caustic soda, soda Vash, ammonia, "amines and the like; however, va Vvolatile agent, such Aas aqueous ammoniais particularly Ipreferred. A.-

Y moni'a may be removed -irom the product 'byVV vacuum drying. It also may be recycled with the falcchol :in Vthe extractionv process.A

The amount of'allrali, or alkaline agent, to be used -will vary, 3'but in Vgeneral fit is such aswill not raise the pH of the casein aboveta `poi'ntfwhere it loses lits granular form.' *In general this per mits the' use' of a treating f'solut'ion having 'an initial pH o'f '7 Ito H9."v Extraction "i's'substantially complete when the casein has been 'treated with from 5 to 15y parts kbyvolume .o'f alcohol per part of fnase'in b3' buik "voiume, The extraction ,teme

perature is preferably 'within the :range Aiofnbout vAnfapparatus-'vias usedcons'isting "oil ani ex-f tractor, an extractphaseboiler; and-'a solvent condenser, so farrangedfand connectedfthat the soll', vent `vapors"-Yp'a'ssedi `from lvtirebil'er to* ftlnel'con-v denser, thefccndensate iiowcd Tby'gravity to "the extractor, and: the extract iiowedf br-gravity 'from the extractor to the boiler. 150 'pounds of dry granular, commercial casein was charged to the apparatus 'together with v350 V"liters of methanol. The casein Wasi treated Vwith the methanol at," about 51" F. blowthe boiling 'temperature of 'the latter :for `about lhour; vThe casein'wasconta'ined in ya sturdyifcloth bag `in the extractor. y"The -rnethanol was boiled inthe boiler, the -vapors wereA condensed, the lcondensate was `passed through" the casein, andthe fextract phase was reboiled after Tit :waswithdrawn from-the extractor. Then an alkaline agent consisting of 30G cc. of 284% l"arninonia water dissolved in `1 gallon of methanol l was gradually yadded through jan inlet in the top of the extractorfover aperiod of 11/2` hours, While the methanol circulation treatment was -con tinued. 'The alkaline methanol 'WaSthen circulated through 'the system for vincurs. The pH oftheV alkaline solution varied vfrom substantially 7 'to .9- #at the start of the alkaline treat-I` ment to about l61" to "7.5 at v'the end of the ex-A traction. V"lfhe process Wastermlnatedby draw-" Aing oiT all the extractrfromvthe Aextractor"and VshuttingV olf fthe 1heatisupplied to the boiler. The,

casein removed from the vextractor vand dried i in a vacuum ovenrat abbutlf" F. The casein product obtained had anY excellent appearance.

It was in :the rom: of slightly yeimwish white' granules of uniform color. This product was compared with a commercial so-called devitaminized casein, for vitamin content. The respective analyses follow:

f control in basal feeding diets.

Commercial Product o! Vitamin Ugfd "vitaminthe above ex- Free Cassin ample Cassin Riboavin gamma per Ngm. casein 8. 0 1. 5 0. 9

iacin emma per casein? 2.2 1.1 0.5 Pantothenic Acid, gammaper gm.casei.n 3.4 1.1 0.4

The extract phase contained the crude extracted vitamins. The vitamins may be recovered therefrom by known methods. Thel vitamins so recovered are suitable for the usual vitamin uses.

If` desired, the pH of the nal casein product may be adjusted to its iso-electric point by the addition of small amounts of dilute acid, such as acetic, lactic or hydrochloric acid, prior todrying. This may be done by circulating a methanol solution ofthe acid throughthe casein, in the system used for the extraction, or af similar system, after the extraction is complete. However, for most purposes, this will not be necessary, The pH of the casein obtained by the process is usually not over one unit above the iso-electric point. i

It may be desirable to soak and swell the casein in cold or hot solvent prior toits treatment in the extractor.

The vitamin content of the product prepared in accordance with the invention is much lower than that of the commercial so-called vitamin free caseins. This makes possible much better The term vitamin-free, as used herein,is intended to indicate absence of the vitamins which-are now known.

In view of the foregoing variations and modications of the disclosed steps, treating agents, conditions, and the` like will be apparent to those skilled in the art. The invention includes all such variation-s and modiiications as fall within the scope of the appended claims.

We claim:

1. A process of separating vitamins from casein containing vitamins which comprises adjusting and maintaining said casein at a pH in the l range of 4.7 to 6.0 by treating it with an alkaline methanol solution to form an extract-phase, recovering substantially vitamin free` casein, and recovering the vitamins originally in the casein `charge from the extract phase.

methanol solution having an initial pH in the rangeof about 7 to about 9 to form an extract phase, recovering substantially vitamin free casein, and recovering the vitamins originally "in the casein charge from the extract phase.

3. A process of separating vitamins from casein containing vitamins which comprises adjusting and maintaining said casein at a pH in the range of 4.7 to 6.0 by treating it with ammoniacal methanol having an initial pH in the range of about 7 to about9 to form an extract phase, recovering substantially vitamin free casein, and recovering the vitamins originally in the casein charge from the extract phase.

4. A process of separating vitamins from casein containing vitamins which comprises adjusting and maintaining said casein in a granular form at a pH in the range of 4.7 to 6.0 by treating it with an alkaline methanol solution to form an extract phase, and recovering substantially vitamin free casein by separating the treated casein in the form of individual particles from said extract phase.

5. A process of separating vitamins from casein containing vitamins which comprises adjusting and maintaining said casein in a granular form at a pH in the range of 4.7 to 6.0 by treating it with an alkaline methanol solution having an initial pH in the range of about 7 to about 9 to form an extract phase, and recovering substantially vitamin free casein by separating the treated casein in the form of individual particles from said extract phase.

6. A process of separating vitamins from casein containing vitamins which comprises adjusting and maintaining said casein in a granular form at a pH in the range of 4.7 to '6.0 by treating it with ammoniacal methanol having an initial pH in the range of about 7 to about 9 to form an extract phase, and recovering substantially vitamin free casein by separating` the treated casein in the form of individual particles from said extract phase.

l SAMUEL M. WEISBERG.

JOSEPH GREENSPAN.

REFERENCES CITED The following references are of record in th file of this patent: l

bNrrEn s'rA'rEs PA'I'EN'rs Number A Name Date 2,052,218 Dickens Aug. 25, 1936 2,202,307 Booher May 28, 1940 n OTHER REFERENCES Day, Journal of Biological Chemistry, vol. 119, page xxii (19137).

Maitra, Biochemical Journal, v ol. 33, pages 164 to 1651 (1939).

U. S. Dispensatory, 22nd ed. (1937), page 1684. 

